ANALYSIS OF OLIGONUCLEOTIDE IMPURITIES ON AN UHPLC-TOF MS SYSTEM WITH A MODIFIED SURFACE TECHNOLOGY

Význam tématu

Oligonucleotide therapeutics represent a rapidly growing class of drugs that demand highly selective and sensitive analytical methods for impurity identification and quantification. Conventional UHPLC systems suffer from nonspecific adsorption of negatively charged oligonucleotides on metal surfaces, leading to analyte loss, distorted peak shapes, and poor reproducibility.

Cíle a přehled studie / článku

This work evaluates the performance of the ACQUITY Premier UPLC BioAccord™ LC-MS platform, which integrates MaxPeak High Performance Surfaces (HPS) technology and a Premier OST column, for intact mass confirmation and quantitation of low-level oligonucleotide impurities. The goal is to demonstrate reduced surface interactions, reliable carryover elimination, and broad dynamic range.

Použitá metodika a instrumentace

• LC: Ion-pair reversed-phase separation on two 2.1×100 mm C18 columns (Premier OST vs. conventional), using Solvent A (7 mM TEA, 40 mM HFIP in water) and Solvent B (3.5 mM TEA, 20 mM HFIP in 50 % methanol). A 25 min gradient from 25 to 35 % B at 0.3 mL/min and 60 °C.
• MS: BioAccord LC-MS System with ACQUITY Premier fluidics incorporating MaxPeak HPS, operated in negative ESI mode, TOF detection over m/z 400–5000 at 1 Hz scan rate.
• Data processing: waters_connect™ software with Bayesian charge deconvolution.

Hlavní výsledky a diskuse

• MaxPeak HPS and OST column significantly decreased nonspecific binding, yielding sharp, symmetrical peaks and excellent run-to-run reproducibility.
• Carryover evaluation after 1000 nM injections showed no detectable oligonucleotide signal in blank runs.
• Calibration curves demonstrated linearity (R2>0.99) over three orders of magnitude (0.5–1000 nM), with mass accuracy < 15 ppm for all components.
• Low-abundance impurities down to 0.2 % relative level were reliably detected and quantified.

Přínosy a praktické využití metody

The inert UHPLC-TOF MS workflow enhances QC and R&D of oligonucleotide therapeutics by providing intact mass confirmation, sensitive impurity profiling, and robust quantitative performance. Laboratories benefit from reduced analytical variability and streamlined data processing.

Budoucí trendy a možnosti využití

Future developments may include expanding inert surface chemistries to microflow and nanoLC formats, integration with high-throughput automated platforms, and incorporation of AI-driven deconvolution algorithms for comprehensive impurity characterization.

Závěr

Implementation of MaxPeak HPS technology within the ACQUITY Premier UPLC BioAccord system and Premier OST columns effectively mitigates surface interactions, eliminates carryover, and enables accurate, sensitive analysis of oligonucleotide impurities across a broad concentration range.

Reference

• Sharma VK, Watts JK. Oligonucleotide therapeutics: chemistry, delivery and clinical progress. Future Med Chem. 2015;7(16):2221-2242.
• DeLano M, Walter T, Lauber M, et al. Mitigate analyte interactions with metal surfaces in UHPLC. Anal Chem. 2021;93:5773-5781.
• Doneanu C, Knowles C, Fox J, et al. Analysis of oligonucleotide impurities on the BioAccord system with ACQUITY Premier. Application Note 720007301, Waters Corporation; 2021.